The Transcreener IKK-beta Assay determines IKK-beta enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener IKK-beta Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The IKK-beta assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The adenosine kinase assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized IKK-beta reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ shown here = 0.87.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use both 25 µM ATP with 15 µM IKKTide peptide substrates. Non-productive hydrolysis of ATP occurs in many kinase reactions due to many factors including autophosphorylation and water exploiting the ATP binding pocket of the enzyme.
Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 7.5 nM IKK beta, 10 µM ATP, 10 µM IKKtide for an enzyme reaction of 60 minutes at room temperatur
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
Assay conditions for the IKK-beta titration shown include 25 µM ATP, 15 µM IKKtide, and 24 µg/mL ADP² antibody.
Assay conditions for the IKK-beta titration shown include 25 µM ATP, 15 µM IKKtide, 55.3 nM tracer, and 8 nM ADP² Tb Antibody.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for FP 28 μg/mL for 25 μM ATP. Yours may vary depending on, enzyme, and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Stops enzyme reactions requiring Mg²+. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 25 μM for IKK-beta. |
| Component | Notes |
|---|---|
| IKK-beta Enzyme | We have successfully used recombinant full-length human IKK-beta from ThermoFisher Cat. #PV3836. We've used IKK-beta at 7.5 nM for screening purposes. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 5 mM MgCl2,, 0.01% Brij-35. Enzyme buffer components may be optimized as required. |
| Substrate | Use the substrate to be phosphorylated for your experiment. Here we used IKKtide at 15 µM (SignalChem Cat. #133-58) |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
IKK-beta is a serine kinase in the NF-κB pathway. IKK aids in activiating NF-κB by phosphorylating a bound inhibitor IκBα, allowing NF-κB to enter the nucleus, in turn, activating various inflammatory responses. The signaling pathway is part of the innate immune response to bacterial and viral material, among other stimuli.
Incorrect regulation can lead to a number of conditions including inflammatory autoimmune disorders and cancer. Because of this, IKK-beta modulators have become emerging targets in drug discovery.
The Transcreener ADP2 IKK-beta Assay is an excellent tool for researchers examining the therapeutic effects of IKK-beta.
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